The potential of utilizing exosomes as a therapeutic delivery system for biological and chemical drugs is currently a highly active area of clinical research. However, the field is currently facing challenges such as low yield and heterogenicity of exosomes produced by mammalian cells. To address these issues, Mimetic-Nano Vesicles (M-NVs) are an emerging and potentially promising alternative therapeutic delivery system to exosomes. M-NVs can be generated from all types of cell lines using a systematic purification protocol with advantages such as, reproducibility, large scale production, uniformity and cost effectiveness. In this study, M-NVs were generated by extruding Human neuroblastoma SH-SY5Y cells (n = 5) using an Avanti mini-extruder and purified by OptiPrep™ density gradients. Generated M-NVs were comprehensively characterized based on biophysical characteristic and further compared to exosomes. Our protocol was able to reproducibility demonstrate the production of M-NVs at high-yield (>100-fold) when compared to endogenously secreted exosomes from SH-SY5Y cells. Proteomic analysis indicated the presence of key exosomal membrane surface features on M-NVs. RNA deep sequencing revealed that M-NVs cargo closely mirrored parental cells however, endogenous exosomes were found to specifically package certain RNA species. In summary, results from this study demonstrated a reproducible large-scale production of M-NVs, provided key insights into M-NVs cargo and highlighted its potential as an alternative to exosomes for therapeutic delivery system.